A method for multi-layer in-situ soil sampling suitable for environmental microbial research

Abstract

The method comprises the following steps: a modular sampling device drives a sampling drill bit carrying a sterile thin-walled sampling cylinder to a preset depth of the ground for penetration through a built-in depth travel encoder and a torque sensor; the collected columnar soil core is divided into independent discrete sample blocks in an in-situ state, and then the discrete sample blocks are sequentially transported to the feed port of a pretreatment module; a weighing sensor at the bottom of a micro processing cavity measures the instantaneous mass of the obtained soil sample, a precision micro pump system extracts a specific volume of sterile nucleic acid protection liquid from a pre-storage liquid tank and injects it into the micro processing cavity, and homogenization treatment is performed; the homogenized soil slurry is filled into a sterile sampling tube with a bar code identification label; and the sealed sterile sampling tube is transferred to a refrigerated storage box for storage. Through cooperation of the modular sampling device and the pretreatment module, the standardization degree of sample processing can be increased, and the repeatability of test data can be improved.

Description

Technical Field

[0001] This invention belongs to the field of soil sampling technology, and in particular relates to a multi-layer in-situ soil sampling method suitable for environmental microbiology research. Background Technology

[0002] Traditional soil sampling typically involves physically cutting soil cores in the field to create stratified samples, which are then placed in sampling bags or centrifuge tubes and transported using vehicle-mounted ice packs or liquid nitrogen tanks. While this discrete approach of "field collection, laboratory pretreatment" is feasible for obtaining macroscopic samples, it still has some shortcomings as environmental microbiome research shifts towards greater precision. (1) Soil microorganisms are highly sensitive to changes in the microenvironment. Microbial groups at different depths have adapted to specific dissolved oxygen gradients, nutrient levels, and stress environments over a long period of time. Once the sampling equipment disrupts the physical structure of the in-situ habitat and exposes it to the atmospheric environment, the metabolic homeostasis inside the soil will rapidly collapse. During the time gap between the sample being taken out of the in-situ and entering the stabilization treatment, microorganisms will produce a violent stress response, resulting in a large-scale reconstruction of intracellular RNA transcription levels within seconds to minutes. In addition, DNA degradation enzymes will also have increased activity due to drastic environmental changes, leading to a deviation between the multi-omics data measured in the laboratory and the actual in-situ state of the soil.

[0003] (2) Traditional sampling methods can easily lead to the migration of highly abundant microorganisms on the surface with the drilling tools due to the collapse of the soil structure, thus contaminating the deep samples. This vertical material disturbance significantly interferes with the study of the vertical distribution of microbial communities, especially the microevolution at the junction of the anaerobic and aerobic zones.

[0004] (3) In the simple working environment in the field, the manual weighing, homogenization and addition of nucleic acid protection solution of samples is not only extremely labor-intensive and inefficient, but also difficult to guarantee the accuracy of the liquid addition ratio and the uniformity of mixing. In addition, the environmental exposure during the operation will introduce interference from non-target biological substances, further reducing the signal-to-noise ratio of the sample.

[0005] Therefore, designing a method that integrates precise multilayer sampling, real-time preprocessing, and standardized nucleic acid homeostasis maintenance to avoid cross-contamination and environmental interference has significant practical implications. Summary of the Invention

[0006] The purpose of this invention is to provide a multi-layer in-situ soil sampling method suitable for environmental microbiology research. By combining a modular sampling device and a pretreatment module, it solves the problem that deep soil samples are easily contaminated by surface biological signals, and can ensure a high degree of standardization in sample processing and reproducibility of experimental data.

[0007] To achieve the above objectives, this invention provides a multi-layer in-situ soil sampling method suitable for environmental microbiology research, comprising the following steps: S1. The modular sampling device drives the sampling drill bit, which is equipped with a sterile thin-walled sampling tube, to penetrate to a preset depth underground through the built-in depth stroke encoder and torque sensor. The preset depth is divided into multiple continuous sampling intervals from shallow to deep. In each sampling interval, the sampling drill bit uses a constant axial feed rate to cut and sample in order to maintain the in-situ physical structure of the soil core. S2. After completing the penetration of the preset depth range, the sample core is cut at each layer interface of the sterile thin-walled sampling tube to divide the collected columnar soil core into independent discrete sample blocks in situ. Then, each discrete sample block is transported to the feed port of the pretreatment module in sequence. S3. After receiving the discrete sample block, the preprocessing module places it into the corresponding micro-processing chamber. The weighing sensor at the bottom of the micro-processing chamber performs instantaneous mass measurement of the acquired soil sample. According to the preset liquid-solid ratio parameter, the controller drives the precision micro-pump system to extract a specific volume of sterile nucleic acid protection solution from the pre-storage liquid tank and inject it into the micro-processing chamber. After injection, the planetary ball mill homogenizing mechanism in the micro-processing chamber is activated to perform high-frequency homogenization of the soil sample and sterile nucleic acid protection solution in a closed environment. This allows the sterile nucleic acid protection solution to fully penetrate into the soil aggregates, instantly blocking the metabolic activity of microorganisms and the catalytic effect of nucleic acid degradation enzymes at the molecular level. S4. The homogenized soil slurry is filled into sterile sampling tubes with barcode identification labels and sealed in a sealing bag; S5. Transfer the sealed sterile sampling tube to a refrigerated storage box. The refrigerated storage box uses semiconductor refrigeration technology to maintain the internal environment at a constant temperature until the field sampling mission is completed.

[0008] The above-mentioned soil multilayer in-situ sampling method suitable for environmental microbiology research, wherein the sampling drill bit of the modular sampling device is made of high-strength alloy steel and the surface is treated with diamond-like carbon coating; the sterile thin-walled sampling tube is lined with a medical-grade polytetrafluoroethylene self-sealing film, which is hydrophobic and bio-inert.

[0009] The above-mentioned soil multilayer in-situ sampling method suitable for environmental microbiology research, wherein, in step 3, the precision micropump system employs a piezoelectric ceramic-driven microfluidic pump, and the injection volume of sterile nucleic acid protection solution is... The calculation formula is: in, The weight of the soil sample measured by the weighing sensor. The density of the sterile nucleic acid protection solution is... The dilution factor is a pre-defined dilution factor based on the soil's bulk density and porosity. It is 3-5.

[0010] The above-mentioned soil multilayer in-situ sampling method suitable for environmental microbiology research, wherein the chemical composition of the sterile nucleic acid protection solution includes saturated concentrations of ammonium salts, chelating agents, and protein denaturing agents.

[0011] The above-mentioned soil multilayer in-situ sampling method suitable for environmental microbiology research is provided in which the bottom of the sterile sampling tube is equipped with an RFID radio frequency identification chip. The controller automatically writes multidimensional metadata, including sampling coordinates, depth, soil sample quality, protective liquid ratio and storage temperature, into the RFID radio frequency identification chip, thereby realizing a closed-loop data system and traceability throughout the entire process from in-situ collection to subsequent analysis.

[0012] Compared with the prior art, the present invention has the following advantages: (1) By combining modular sampling devices and pretreatment modules, the time difference between physical acquisition and biochemical fixation in traditional sampling processes can be solved, which has a significant effect on studying unstable mRNA expression profiles and metabolic intermediates with extremely short half-lives in soil.

[0013] (2) By using precise physical segmentation and self-sealing lining, the problem of deep soil samples being easily contaminated by surface biological signals is solved by traditional excavation sampling methods, laying a good foundation for studying the evolution of soil microbial communities with vertical physicochemical gradients.

[0014] (3) By replacing tedious manual field operations with mechanized operations, the efficiency of field operations is improved, and to a certain extent, systematic interference caused by differences in personnel operation, environmental exposure pollution and weighing errors is eliminated, ensuring a high degree of standardization of sample processing and repeatability of experimental data. Attached Figure Description

[0015] Figure 1 This is a schematic flowchart of the method of the present invention; Figure 2 This is a schematic diagram of the principle structure of the system of the present invention; Figure 3 This is a schematic diagram of the preprocessing module of the present invention; Figure 4 This is a schematic diagram of the structure of the refrigeration storage box of the present invention; Figure 5 This is a schematic diagram of the overall structure of the device of the present invention; Figure 6 This is a schematic diagram of the modular sampling device of the present invention; Figure 7 This is a schematic diagram of the preprocessing module of the present invention; Figure 8 This is a bottom view of the preprocessing module of the present invention; In the picture: 1. Modular sampling device; 11. Servo motor; 12. Sampling drill bit; 2. Pre-treatment module; 21. Drive motor; 22. Precision micro-pump system; 23. Feed inlet; 24. Micro-processing chamber; 25. Conveying valve; 26. Mounting bracket; 3. Refrigerated storage box; 4. Mobile vehicle. Implementation To further illustrate the technical means and effects adopted by the present invention in order to achieve the intended purpose, the specific embodiments according to the present invention will be described in detail below with reference to the accompanying drawings and preferred embodiments.

[0016] Please see Figure 1-8 This invention provides a multi-layer in-situ soil sampling method suitable for environmental microbiology research. The entire sampling equipment consists of a modular sampling device 1, a pretreatment module 2, and a refrigerated storage tank 3. All components are mounted on a mobile vehicle 4 and interact with each other via a controller. The number of pretreatment modules 2 can be set on the mobile vehicle 4 according to soil collection needs. Specifically, the entire pretreatment module is fixed to the mobile vehicle 4 by the mounting bracket 26 at the lower end of the micro-processing chamber, and then the inlet of the precision micropump system 22 is connected to an external pre-storage liquid tank.

[0017] The core components of the modular sampling device 1 are a sampling drill bit 12 with high-strength cutting capabilities and a matching sterile thin-walled sampling tube. The sampling drill bit 12 uses high-strength alloy steel as its base, and its surface is coated with diamond-like carbon (DLC). When the sampling drill bit 12 penetrates hard soil, it can suppress the instantaneous temperature rise caused by the intense friction between the drill bit and the soil, avoiding thermal stress reactions and nucleic acid degradation induced by thermal disturbance in microorganisms. The sampling drill bit 12 has a hollow channel inside to accommodate the sterile thin-walled sampling tube. The wall thickness of the sterile thin-walled sampling tube is 0.8-1.2 mm to reduce the compressive stress on the soil core. Furthermore, the entire inner surface of the sterile thin-walled sampling tube is lined with a medical-grade polytetrafluoroethylene (PTFE) self-sealing film. This film has extremely high hydrophobicity and bioinertness, ensuring that no shallow soil residue adheres to the deep soil core during the process of entering the sterile thin-walled sampling tube.

[0018] During the sampling execution phase, the modular sampling device 1 drives the sampling drill bit 12 downward through the servo motor 11. In order to achieve precise control of the sampling depth, a multi-turn absolute depth stroke encoder is integrated at the spindle position. At the same time, the torque sensor monitors the torque feedback received by the sampling drill bit 12 during the cutting process in real time and adjusts the torque of the servo motor 11. When encountering high-density soil layers, the axial feed rate is automatically reduced to maintain a constant cutting energy density.

[0019] After sampling, the columnar soil core collected in the sterile thin-walled sampling tube is divided into independent discrete sample blocks in situ using a cutting tool. The separated discrete sample blocks are then removed from the sterile thin-walled sampling tube and placed into the feed inlet 23 of the corresponding pretreatment module. Once the sample block is placed into the microprocessing chamber 24, the weight signal m from the sample position sensor installed at the bottom of the chamber is measured. soil The data is transmitted via cable to the controller, which calculates the required volume of nucleic acid protection solution to be injected in real time based on preset liquid-to-solid ratio parameters. The calculation formula is: in, The weight of the soil sample measured by the weighing sensor. The density of the sterile nucleic acid protection solution is... The dilution factor is a pre-defined dilution factor based on the soil's bulk density and porosity. The value is 3-5. After receiving the instruction, the precision micropump system 22 draws a precise volume of protective liquid from the pre-stored liquid tank.

[0020] The nucleic acid protection solution used in this invention is a specially formulated composite solution, the main components of which include saturated ammonium sulfate, 0.5M ethylenediaminetetraacetic acid (EDTA), and 1% sodium dodecyl sulfate (SDS). Ammonium sulfate alters the ionic strength of the solution, causing soil proteins (including various nucleases) to precipitate and become inactive; EDTA efficiently chelates metal ions such as Mg²⁺ and Ca²⁺, blocking the catalytic activity of metal ion-dependent deoxyribonuclease (DNase) and ribonuclease (RNase); and SDS, as a surfactant, assists the protection solution in penetrating through soil aggregates.

[0021] To further accelerate the infiltration process, a planetary ball mill homogenizing mechanism is installed within the micro-treatment chamber 24. This mechanism, activated by a drive motor 21, contains several zirconia homogenizing beads with a diameter of 2-3 mm. During operation, the planetary ball mill homogenizing mechanism adopts a dual-axis rotation mode, with its revolution speed set at 300-400 rpm and its rotation speed at 600-800 rpm, maintaining a fixed revolution-to-rotation ratio of 1:2. Under this high-frequency oscillation environment, soil aggregates are thoroughly broken down into a slurry state with a particle size of less than 2 mm in a short time. Through this simultaneous action of "mechanical breaking + chemical fixation," the protective liquid can quickly come into contact with each microbial cell, thereby fixing the metabolic profile of the microorganisms at the moment they leave the soil.

[0022] After homogenization, the soil slurry is filled through delivery valve 25 into sterile sampling tubes equipped with barcodes and RFID chips. These tubes are then placed within the internal array of the refrigerated storage chamber 3. The internal space of the refrigerated storage chamber 3 is maintained at a constant temperature of 4℃ ± 0.5℃. The temperature control system, based on a PID algorithm, dynamically adjusts the current power of the semiconductor cooling chip by using four platinum resistance temperature sensors distributed around the chamber to provide real-time feedback of the temperature field data. Upon arrival at the storage chamber, the RFID chip is written with comprehensive metadata for the sample, including precise sampling latitude and longitude, sampling depth, weight, protective liquid ratio, homogenization time, and the entire temperature curve, providing a complete data chain for subsequent multi-omics analysis in the laboratory.

[0023] To ensure sterility during each sampling operation, the sampling probe is fully irradiated with a high-intensity ultraviolet (UVC) lamp at a wavelength of 254 nm during each sampling, with an irradiation dose of no less than 30 mJ / cm². Simultaneously, a hydrogen peroxide spray device sprays a 3% hydrogen peroxide mist into the sampling drill bit 12 and the interior of the sterile thin-walled sampling tube, using strong oxidation to remove any remaining biological DNA / RNA molecules, or a brand-new sterile thin-walled sampling tube is used.

[0024] In addition, regarding spatial resolution, the servo drive system with a depth stroke encoder can control the actual sampling depth error to millimeter precision. In contrast, traditional methods are limited by manual visual inspection and manual cutting, resulting in a high rate of cross-contamination between layers. When processing deep samples with extremely low biomass, this level of contamination can mask the true layer characteristic signals.

[0025] When the planetary ball mill homogenizing mechanism rotates at high speed, the homogenizing beads generate powerful shear forces and instantaneous impact forces on soil aggregates. According to Hertzian contact theory, the instantaneous impact pressure of a 2mm diameter zirconia bead at 800rpm can reach several gigapascals (GPa), sufficient to break down the common silicate mineral framework and micron-sized microbial capsules in the soil. Because the treatment is carried out in a sealed chamber filled with nucleic acid protective solution, every tiny soil particle produced by the breakup is immediately surrounded by the protective solution, thus achieving instantaneous fixation at the molecular level.

[0026] The method disclosed in this embodiment is not only applicable to soil sampling with low moisture content such as black soil and sandy soil, but also has good applicability to swamp soil with high moisture content or calcareous soil with high hardness. When sampling high moisture content soil, the controller will automatically adjust K based on the low resistance characteristics fed back by the torque sensor. ratio The coefficient is adjusted to the upper limit, and the running time of the planetary ball mill homogenizing mechanism is appropriately extended to compensate for the dilution effect of water on the concentration of the protective solution. When dealing with high-hardness soils, the micro-impact mode of the sampling drill bit 12 is activated, using high-frequency vibration to assist cutting and protect the sampling tube from deformation.

[0027] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A multi-layer in-situ soil sampling method suitable for environmental microbiology research, characterized in that, Includes the following steps: S1. The modular sampling device drives the sampling drill bit, which is equipped with a sterile thin-walled sampling tube, to penetrate to a preset depth underground through the built-in depth stroke encoder and torque sensor. The preset depth is divided into multiple continuous sampling intervals from shallow to deep. In each sampling interval, the sampling drill bit uses a constant axial feed rate to cut and sample in order to maintain the in-situ physical structure of the soil core. S2. After completing the penetration of the preset depth range, the sample core is cut at each layer interface of the sterile thin-walled sampling tube to divide the collected columnar soil core into independent discrete sample blocks in situ. Then, each discrete sample block is transported to the feed port of the pretreatment module in sequence. S3. After receiving the discrete sample block, the preprocessing module places it into the corresponding micro-processing chamber. The weighing sensor at the bottom of the micro-processing chamber performs instantaneous mass measurement of the acquired soil sample. According to the preset liquid-solid ratio parameter, the controller drives the precision micro-pump system to extract a specific volume of sterile nucleic acid protection solution from the pre-storage liquid tank and inject it into the micro-processing chamber. After injection, the planetary ball mill homogenizing mechanism in the micro-processing chamber is activated to perform high-frequency homogenization of the soil sample and sterile nucleic acid protection solution in a closed environment. This allows the sterile nucleic acid protection solution to fully penetrate into the soil aggregates, instantly blocking the metabolic activity of microorganisms and the catalytic effect of nucleic acid degradation enzymes at the molecular level. S4. The homogenized soil slurry is filled into sterile sampling tubes with barcode identification labels and sealed in a sealing bag; S5. Transfer the sealed sterile sampling tube to a refrigerated storage box. The refrigerated storage box uses semiconductor refrigeration technology to maintain the internal environment at a constant temperature until the field sampling mission is completed.

2. The soil multilayer in-situ sampling method suitable for environmental microbiology research according to claim 1, characterized in that, The sampling drill bit of the modular sampling device is made of high-strength alloy steel and its surface is treated with diamond-like carbon coating; the inside of the sterile thin-walled sampling tube is lined with a medical-grade polytetrafluoroethylene self-sealing film, which is hydrophobic and bio-inert.

3. The soil multilayer in-situ sampling method suitable for environmental microbiology research according to claim 1, characterized in that, In step 3, the precision micropump system employs a piezoelectric ceramic-driven microfluidic pump, and the injection volume of the sterile nucleic acid protection solution is... The calculation formula is: in, The weight of the soil sample measured by the weighing sensor. The density of the sterile nucleic acid protection solution is... The dilution factor is a pre-set dilution factor based on the soil's bulk density and porosity. It is 3-5.

4. The soil multilayer in-situ sampling method suitable for environmental microbiology research according to claim 1, characterized in that, The chemical composition of the sterile nucleic acid protection solution includes saturated concentrations of ammonium salts, chelating agents, and protein denaturants.

5. The soil multilayer in-situ sampling method suitable for environmental microbiology research according to claim 1, characterized in that, The bottom of the sterile sampling tube is equipped with an RFID radio frequency identification chip. The controller automatically writes multidimensional metadata, including sampling coordinates, depth, soil sample quality, protective liquid ratio, and storage temperature, into the RFID radio frequency identification chip, realizing a closed-loop data system and traceability throughout the entire process from in-situ collection to subsequent analysis.

Images